PROJECT SUMMARY Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), leads to 9 million new cases and 1.5 million deaths every year. Mtb can be internalized by macrophages through phagocytosis, escape the macrophage defense mechanism, and then resist inside macrophages (majorly in alveolar macrophages). Macrophage reservoirs significantly reduce the drug efficacy; thus, TB therapy requires continuous and frequent drug dosing with a minimum 6 month-treatment. The long duration of the therapy often leads to incomplete adherence to anti- TB treatment, resulting in the development of drug resistance. The emergence of multidrug and extensively drug resistant TB (M.XDR-TB) has become a major public health threat. It has been shown that macrophage-targeting liposomal anti-TB drugs can increase local drug concentrations inside macrophages and eventually enhance drug efficacy. However, in contrast to other types of drug delivery systems (e.g. antibody-based drug delivery in cancer therapy), the existing anti-TB drug delivery systems are limited by several factors. First, the frequently used molecules for targeting macrophages are not the best targeting ligands. To find the best binding partners, a comprehensive ligand screening should be performed, but such screening has rarely been conducted. Second, the current anti-TB drug deliveries majorly used glycans as ligands to target macrophage glycan binding receptors. Glycans are a good choice for targeted drug delivery because these molecules are biocompatible, less immunogenic, small enough to cross tissue barriers, and relatively inexpensive. However, glycan-protein interactions are typically weaker than the antibody-antigen interactions, leading to poor targeting efficiency. To address these issues, the PIs propose to design a novel liposomal anti-TB drug carrier to target macrophages. This new drug carrier possesses several unique features. First, to enhance the affinity between glycan ligands and macrophages, the hetero-multivalent targeting strategy is employed, wherein multiple ligands on a single liposome simultaneously bind to multiple different receptors on a single macrophage. Second, liposomes are chosen as drug carriers because liposomes can offer the unique two-dimensional membrane fluidity that accelerates the hetero-multivalent binding process. Third, to find the best binding ligands for targeting macrophage receptors, the PIs will use our special high-throughput nanocube-based array to screen the ligand library. Ultimately, the PIs will encapsulate rifampicin into the optimized hetero-multivalent targeted liposome and evaluate its antimicrobial activity against intracellular Mtb. The success of this study will lead to a new liposomal carrier which encapsulates multiple antibiotics to effectively treat resistant strains.